Method for producing DNA vectors from molecular bricks containing sequences of interest

A method for producing a circular double-stranded DNA vector comprising at least six sequences of interest, said method consisting of:

• (a) a step of simultaneously contacting at equimolar ratio at least six molecular building blocks, which are different from one another, in the presence of a single type IIs restriction enzyme and a ligase,
• each molecular building block consisting of an isolated linear double-stranded DNA molecule and containing:
• (i) a sequence of interest with no specific recognition site of the aforementioned type IIs restriction enzyme,
• (ii) two double-stranded DNA adapters, flanked upstream and downstream of said sequence of interest, each double-stranded DNA adapter comprising a sequence of at least 12 nucleotides, which sequence contains:
• a single and only recognition site of the aforementioned type IIs restriction enzyme,
• the recognition site of the aforementioned type IIs restriction enzyme of the adapter upstream of said sequence of interest and the recognition site of the aforementioned type IIs restriction enzyme of the adapter downstream of said specific sequence being convergent,
• (b) a step of incubation of the mixture obtained in step a) at a temperature ranging from 20° C. to a temperature of 55° C., during a period ranging from 2 minutes to a period of 30 minutes, which step leads:
• to the elimination by cleaving of the recognition sites of the type IIs restriction enzyme used,
• to the formation of a cohesive single-stranded suture of at least 2 nucleotides at each of the ends of said sequence of interest,
• said cohesive single-stranded suture of at least 2 nucleotides upstream of one of the at least six sequences of interest being complementary to said cohesive single-stranded suture of at least 2 nucleotides downstream of another sequence of interest,
• to the pairing by nucleotide complementarity of the aforementioned cohesive single-stranded sutures of at least 2 nucleotides, and
• to the positioning of the sequences of interest contiguously with one another in an order and a single and defined direction,
• said cohesive single-stranded suture of at least 2 nucleotides upstream and downstream of the sequence of interest is selected for assembly using a scoring matrix where each pairing of two sequences of four non-palindromic nucleotides is attributed a score ranging from 0 to 10, where 0 corresponds to a total absence of complementarity (0%) and 10 indicates total complementarity (100%) and each suture selected has a score of complementarity equal to 0, 1 or 2 compared to other sutures selected for assembly,
• (c) a step of ligation of said selected cohesive single-stranded sutures of at least 2 nucleotides, said step being performed at a temperature ranging from 10° C. to a temperature of 40° C. during a period ranging from 2 min to a period of 30 min to obtain a circular double-stranded DNA vector
• said step (b) and (c) being repeated from 1 to 49 times,
• said method also comprising:
• (d) at least one step of incubation at a temperature from 41 to 60° C. during a period ranging from 0.5 to 15 min;
• and
• wherein said steps (a), (b), (c) and (d) are performed in the same reaction mixture.